Citation or identification of any reference in this section or any section of this application shall not be construed as an admission that such reference is available as prior art to the present invention.
An epifluorescence microscope is similar to a conventional reflecting optical microscope in that both microscopes illuminate the sample and produce a magnified image of the sample. An epifluorescence microscope, however, uses the emitted fluorescent light to form an image whereas a conventional reflecting optical microscope uses the scattered illumination light to form an image. The epifluorescent microscope uses a higher intensity illumination, or excitation, light than a conventional microscope. The higher intensity excitation light is needed to excite a fluorescent molecule in the sample thereby causing the fluorescent molecule to emit fluorescent light The excitation light has a higher energy, or shorter wavelength, than the emitted light The epifluorescence microscope uses the emitted light to produce a magnified image of the sample. The advantage of an epifluorescence microscope is that the sample may be prepared such that the fluorescent molecules are preferentially attached to the biological structures of interest thereby producing an image of the biological structures of interest.
A common problem in epifluorescence microscopy is the low contrast, or low signal-to-noise (S/N) ratio, of the fluorescent image. This is due to the low intensity of the emitted light compared to the high intensity of the excitation light. A dichroic mirror is usually used to reduce the scattered excitation light before the image is viewed or recorded.
The dichroic mirror is only partially effective in removing the excitation light from the emitted light so other measures must be taken to increase the S/N ratio of the fluorescent image. In order to assist in the discussion of the other approaches to increasing the S/N ratio of the fluorescent image, reference to FIG. 1 is helpful.
FIG. 1 illustrates the optical path and components of a typical epifluorescence microscope. A sample 100 is placed on a sample holder 105, which is normally a microscope slide. The sample is prepared prior to being placed on the holder 105 with fluorescent tags that bind to the biological structures of interest. The fluorescent tags may be a single type of fluorescent tag that binds to a particular biological structure or may be a mixture of several fluorescent tag types with each tag type binding to a different biological structure. The sample 100 is illuminated by a light source 110 that produces the excitation light with sufficient intensity to cause the tags to emit fluorescent light. The excitation light generated by the light source 110 follows a path 115 through an excitation filter 120 that acts as a band-pass filter allowing only a narrow range of frequencies to pass through the excitation filter 120. The excitation filter 120 is chosen to allow only the light of a frequency that will cause the tags to fluoresce. The excitation light is reflected by a dichroic mirror 130 into the objective lens 140 of the microscope following path 125. A dichroic mirror separates the excitation light from the emission light, in this example, by reflecting the excitation light while transmitting the emission light. The excitation light propagates through the objective lens 140 and illuminates the sample 100 and excites the tags in the sample to emit fluorescent light, also referred to as emission light. The emission light propagates along path 125 in the opposite direction as the excitation light. The emission light passes through the objective lens 140 and through the dichroic mirror 130 and continues along path 135 through an emission filter 150. The emission filter 150 is selected to allow only light matching the frequency of the emission light to pass through the filter. The emission filter 150 may be a band-pass filter, or a long-pass filter that allows the longer wavelength emission light to pass through while stopping the shorter wavelength excitation light. After filtering by the emission filter 150, the emission light is formed into an image by an imaging lens 160.
If the emission filter 150 is perfectly efficient in removing all but the emission light, the magnified fluorescent image would have a very high contrast and S/N ratio. Unfortunately, emission filters are not perfectly efficient so a small amount of excitation light is transmitted though the emission filters. Because the intensity of the excitation light is very high, the small fraction of excitation light that passes through the emission filter is sufficient to severely degrade the contrast of the fluorescent image. In addition, the excitation frequency is usually very close to the emission frequency of the fluorescent tag molecule. The closeness of the two frequencies adds a further requirement on the emission filter that the filter have a very steep adsorption edge between the emission frequency and excitation frequency.
U.S. Pat. No. 6,094,274 issued on Jul. 25, 2000 to Yokoi teaches the use of two interference films as an emission filter. The two interference films act to sharpen the adsorption edge between the emission frequency and excitation frequency. The sharp adsorption edge blocks more of the excitation light while transmitting more of the emission light to the imaging lens.
Another approach to increasing the S/N ratio of a fluorescent image is disclosed in Japanese Application Publication No. 9-292572 by Sudo, et al. published on Nov. 11, 1997 (hereinafter referred to as “Sudo”). Sudo discloses the use of a mirror behind the sample that reflects the excitation light back through the sample. The reflected excitation light approximately doubles the excitation light seen by the sample and therefore approximately doubles the amount of emission light given off by the sample. A portion of the reflected excitation light will, however, also pass through the dichroic mirror and emission filter adding to the “noise” of the higher emission signal. In addition, the increased illumination of the sample from the reflected excitation light increases the bleaching effect on the tagged sample. Bleaching occurs when the fluorescent tag molecules emit decreasing amounts of fluorescent light as the molecules are illuminated by the excitation light. For example, a fluorescent tag molecule will emit less than 10% of its emission intensity after only a minute of being illuminated by the excitation light. As the intensity of the excitation light increases the bleaching rate increases thereby decreasing the emission light and reducing the contrast of the fluorescent image.
Therefore, there still remains a need to provide a microscope system capable of producing a high contrast fluorescent image while reducing unnecessary bleaching of the sample.